CN103217415A - Melamine detection method using a nucleic acid aptamer modified nano-gold particle - Google Patents
Melamine detection method using a nucleic acid aptamer modified nano-gold particle Download PDFInfo
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Abstract
The invention provides a melamine detection method using a nucleic acid aptamer modified nano-gold particle in the field of foodstuff safety technology, and after constructing the detection system with a known melamine concentration, a UV spectrophotometer is employed respectively for determining the ratio of a light absorption value at 640 nm to a light absorption value at 520nm and a standard curve is made. then the object to be measured after pretreatment is added into a mixed liquor containing the melamine aptamer, the nano-gold and PDDA; finally the ratio of the light absorption value at 640 nm to the light absorption value at 520nm is determined, and by contrasting with the standard curve, the concentration of melamine in the object to be measured is obtained. The invention has the advantages of simple operation, rapid visual reaction, and the detection limit reaches to 4.3ppb.
Description
Technical field
What the present invention relates to is a kind of method of food security technical field, specifically is a kind of aptamer modified nanogold particle that utilizes, then the method by the melamine of lowest detectable limit 4.3ppb in naked eyes and the UV spectrophotometer measuring milk.
Background technology
Melamine is a kind of triazines nitrogen heterocyclic ring organic compound, and main application is to be used to produce the melamine formaldehyde resin plastics, heating sublimation, and 300 ℃ of sublimation temperatures, rapid heating and decomposition may the very big prussiate of toxigenicity after the decomposition.Melamine shows alkalescent, can generate melamine salt with various acid reactions.Its nitrogen content is up to 66.6%, and it is a kind of white crystalline powder, do not have what aroma and flavor, is difficult for after the doping being found, if add the 0.1g melamine in 100g milk, just can improve 0.4% protein falseness content, and the name of " extract of protein " is arranged.
The toxicity of melamine own is less, but under the effect of acid or alkali, or in mammalian body and in the bacterium, can be through continuous deammoniation hydrolytic action, be broken down into melamine diamides (ammeline), melamine monamide (ammelide) and cyanuric acid metabolins (seeing Fig. 1-1) such as (cyanuric acid).Wherein melamine and cyanuric acid interact by intermolecular ydrogen bonding, can form water-fast netted association macromolecular complex, cause serious kidney stone.
China national food quality supervision inspection center pointed out on September 13rd, 2008, melamine belongs to industrial chemicals, do not allow to add, October 8 the same year, the Ministry of Public Health, the Ministry of Industry and Information Technology, the Ministry of Agriculture, State Administration for Industry and Commerce and State Administration for Quality Supervision and Inspection and Quarantine unite the issue bulletin, formulate the management value of melamine in breast and dairy products:
1. the value of limiting the quantity of of melamine is 1mg/kg in the baby formula milk powder, and the product that is higher than 1mg/kg must not be sold without exception;
2. the value of limiting the quantity of of melamine is 2.5mg/kg in liquid milk (comprising raw milk), milk powder, other prescription emulsifiable powders, and the product that is higher than 2.5mg/kg must not be sold without exception;
3. the value of limiting the quantity of that contains melamine in breast other consumable products more than 15% is 2.5mg/kg, and the product that is higher than 2.5mg/kg must not be sold without exception.
Detect at melamine at present and mainly contain high performance liquid chromatography, liquid chromatography. mass spectrum/mass spectroscopy etc.In addition, enzyme linked immunosorbent assay, Capillary Electrophoresis, mass spectrum, infrared absorption spectrum, chemiluminescence and electrochemical method also have bibliographical information, these methods generally need be carried out the complicated sample pre-treatment, instrument costs an arm and a leg, detection cost height, and the method complicated operation needs could operate through the personnel of specialized training, can not satisfy the detection needs to melamine residual rapid screening in the milk fully.
Aptamer is by the external evolution of index concentration Fas lignand system (SELEX) technology, filters out the target material is had high specific and high tubercular nucleic acid fragment from a large amount of oligonucleotides storehouse.But compare with biology enzyme with protein-based antibody, aptamer has higher affinity, stability and specificity, and is easy to indicia designs and goes out sensor, has been used for the detection of nucleic acid, albumen, inorganic metal ion and virion and cell.Although can realize that according to prior art aptamers and melamine by hydrogen bonded, still can't utilize the PDDA(PDDA in the prior art) the polymer/nanometer gold grain, and use ultraviolet spectrophotometer to be used for detecting.
Summary of the invention
The present invention is directed to the prior art above shortcomings; a kind of melamine detection method of utilizing the aptamer modified nanogold particle is proposed; can in system, not have under the situation of melamine by specific aptamers; parcel nano Au particle and in conjunction with these characteristics of PDDA; make the adding of melamine compete the aptamers that combines in the system; always make nm of gold lose protection and PDDA reaction; and polymerization; cause the system color from ruby red purple even the blueness of becoming; and system is used for melamine detection in the light absorption value generation marked change at 640nm and 520nm place.
The present invention is achieved by the following technical solutions:
The present invention relates to a kind of implementation method of melamine photosensitivity, by the melamine aptamers is mixed with melamine with nano-Au solution and PDDA solution mixing back successively, the product that obtains after feasible the mixing is in the peak value ratio and the proportional relation of melamine concentration of the resonance scattering signal at 640nm and 520nm wavelength place.
The sequence of described melamine aptamers is: TTTTTTTTTTTTTTTTTTTT, being specially concentration is the ssDNA(single-stranded DNA of 25nM, i.e. single stranded DNA).
The concentration of described nano-Au solution is 55.9 μ g/mL.
The concentration range of melamine is in the product that obtains after the above-mentioned mixing: 0.1 –, 1.0 μ mol/L;
Described mixing specifically is meant: in the centrifuge tube of 5mL, add the ssDNA of 1.0mL25nM and the nano-Au solution of 3.0mL55.9 μ g/mL respectively, after leaving standstill 10 minutes, add the PDDA of 100 μ L0.44nM, in the MOPS damping fluid, hatch after 10 minutes and add melamine.
Described nano-Au solution is meant: citric acid three sodium solutions that add 3.5mL1.0% in the gold chloride of the 0.01wt% that 100mL seethes with excitement, heated and boiled stirred 30 minutes, solution colour becomes shiny red by grey, stops heating and continues to be stirred to cooling, uses 0.2 μ m ultrafiltration membrance filter then.Gained solution is the nm of gold that nanometer particle size is 15nm, and it is standby to place 4 ° of C refrigerators to preserve.
The present invention relates to a kind of melamine detection method of utilizing the aptamer modified nanogold particle, contain the detection architecture of known melamine concentration by structure after, adopt ultraviolet spectrophotometer to measure its ratio and production standard curve respectively at the light absorption value at 640nm and 520nm place; To add through the object to be measured of pre-treatment then and contain in the mixed liquor of melamine aptamers, nm of gold and PDDA; It is last that it obtains the concentration of the melamine in the object to be measured at the ratio of the light absorption value at 640nm and 520nm place by the reference standard curve after measured.
Described pre-treatment is meant: in the centrifuge tube of 10mL, the liquid to be measured of interpolation 5mL1% acetic acid and 1.0mL also left standstill 5 minutes; 10000r is centrifugal 10 minutes then, get supernatant after, regulate pH to 8.0 with 2.0mol/L NaOH, be used for concentration and detect.The effect of pre-treatment is the color and luster influence of removing impurity such as range protein in the milk.
The described detection architecture that contains known melamine concentration is meant: get 10 centrifuge tubes that comprise melamine aptamers, nano-Au solution and PDDA solution, the melamine titer that adds variable concentrations and process pre-treatment respectively, make that content of melamine maintains 0.1-1.0 μ M in the whole detection architecture, measure behind the reaction 10min.
Can detect again after the passing ratio dilution for liquid to be measured far above the detection architecture upper limit; Wherein according to 3 σ/slope, i.e. the ratio of standard deviation and slope, the lowest detection of trying to achieve typical curve is limited to 43nM, can realize by the following extension of typical curve.
Described typical curve is meant: with UV spectrophotometer measuring detection architecture light absorption value A640 and the A520(OD at 640nm and 520nm place), and be horizontal ordinate with the melamine concentration, Δ A=A640/A520 obtains typical curve as ordinate.
Principle of the present invention is: utilize the aptamer can be under the situation that does not have melamine to exist; the parcel nano particle; characteristics in conjunction with PDDA; and the adding of melamine competition bind nucleic acid aptamers; make nano particle lose protection, carry out polymerization under the PDDA effect, the polymerization process solution colour is by ruby red purple even the blueness of gradually becoming; correspondence is that its 620nm place peak value of UV spectrophotometer measuring raises gradually, and 540nm place peak value reduces gradually.And the concentration of melamine is linear in the value of A640/A520 and the certain limit, so change by the signal of analyzing absorbance, can realize melamine detection in the milk.Technique effect
Compared with prior art, the invention has the advantages that easy and simple to handlely, react rapid visual, and lower detectability is arranged.
Description of drawings
Fig. 1 is the principle schematic of aptamer modified nano particle fast detecting melamine.
Fig. 2 is the circular dichroism spectrogram of detection architecture.
Fig. 3 is the influence synoptic diagram of PDDA concentration to detection architecture.
Fig. 4 is the influence synoptic diagram of aptamer concentration to detection architecture.
Fig. 5 is the influence synoptic diagram of reaction time to detection architecture.
Fig. 6 is the synoptic diagram that concerns of melamine concentration and absorbance ratio.
Embodiment
Below embodiments of the invention are elaborated, present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
1) preparation detects liquid: the preparation particle diameter is the nanogold particle of 18nm, and the sequence of aptamer is TTTTTTTTTTTTTTTTTTTT.Preparation detects liquid, in the centrifuge tube of 5mL, adds the ssDNA of 1.0mL25nM and the nano-Au solution of 3.0mL55.9 μ g/mL respectively, leave standstill 10 minutes after, add the PDDA of 100 μ L0.44nM, in the MOPS damping fluid, hatched 10 minutes, obtain detecting liquid.
2) pre-treatment of milk sample: in the centrifuge tube of 10mL, add the milk sample of 5mL1% acetic acid and 1.0mL, left standstill 5 minutes.10000r is centrifugal 10 minutes then, get supernatant after, regulate pH to 8.0 with 2.0mol/L NaOH.
3) detection architecture of the known melamine concentration of preparation: get 10 and added the centrifuge tube that detects liquid, add the variable concentrations melamine titer after the pre-treatment respectively, make that content of melamine maintains 0.1-1.0 μ M in the whole detection architecture, measure behind the reaction 10min.
4) get the standard solution that 200 μ L prepare, place 96 orifice plate plate holes, after the record change color, measure its light absorption value respectively at 640nm and 520nm place with ultraviolet spectrophotometer.640nm and 520nm place light absorption value are respectively A640 and A520, calculate Δ A=A640/A520
5) the peak value ratios delta A with variable concentrations melamine and different wave length maps the drawing standard curve; As shown in Figure 6, this typical curve obtains being y=-0.002+0.883x(R through match
2=0.998).
6) preparation sample system to be measured: after carrying out pre-treatment, mix with detection liquid, fully mixing afterreaction 10min 4 measures its Δ A set by step.
7) gained Δ A per sample looks into typical curve, can be in the hope of content of melamine in the sample.
8) checking: measure three parts with this method and contain that melamine concentration is respectively 0.2,0.5, the milk of 1.0ppm, the recovery that obtains is 90%～120%, has proved the reliability of this method.
9) lowest detection of melamine is limited to 34nM(4.3ppb in this method mensuration milk).The conversion process of nM and ppm is as follows, and 1nM melamine is 10
-9Mol/L melamine, so melamine molecular weight 126 is 1nM=126*10
-9G/L=0.126*10
-3Ppm=0.126ppb.
Claims (10)
1. the implementation method of a melamine photosensitivity, it is characterized in that, by the melamine aptamers is mixed with melamine with nano-Au solution and PDDA solution mixing back successively, the product that obtains after feasible the mixing is in the peak value ratio and the proportional relation of melamine concentration of the resonance scattering signal at 640nm and 520nm wavelength place.
2. method according to claim 1 is characterized in that, the sequence of described melamine aptamers is: TTTTTTTTTTTTTTTTTTTT is specially the single stranded DNA that concentration is 25nM.
3. method according to claim 1 is characterized in that, the concentration of described nano-Au solution is 55.9 μ g/mL; The concentration range of melamine is in the product that obtains after the described mixing: 0.1 –, 1.0 μ mol/L.
4. method according to claim 1, it is characterized in that, described mixing specifically is meant: in the centrifuge tube of 5mL, add the single stranded DNA of 1.0mL25nM and the nano-Au solution of 3.0mL55.9 μ g/mL respectively, after leaving standstill 10 minutes, add the PDDA of 100 μ L0.44nM, in the MOPS damping fluid, hatch after 10 minutes and add melamine.
5. method according to claim 1, it is characterized in that, described nano-Au solution is meant: citric acid three sodium solutions that add 3.5mL1.0% in the gold chloride of the 0.01wt% that 100mL seethes with excitement, heated and boiled stirred 30 minutes, solution colour becomes shiny red by grey, stop heating and continue to be stirred to cooling, use 0.2 μ m ultrafiltration membrance filter then.Gained solution is the nm of gold that nanometer particle size is 15nm, and it is standby to place 4 ° of C refrigerators to preserve.
6. melamine detection method of utilizing the aptamer modified nanogold particle, it is characterized in that, contain the detection architecture of known melamine concentration by structure after, adopt ultraviolet spectrophotometer to measure its ratio and production standard curve respectively at the light absorption value at 640nm and 520nm place; To add through the object to be measured of pre-treatment then and contain in the mixed liquor of melamine aptamers, nm of gold and PDDA; It is last that it obtains the concentration of the melamine in the object to be measured at the ratio of the light absorption value at 640nm and 520nm place by the reference standard curve after measured.
7. method according to claim 6 is characterized in that, described pre-treatment is meant: in the centrifuge tube of 10mL, the liquid to be measured of interpolation 5mL1% acetic acid and 1.0mL also left standstill 5 minutes; 10000r is centrifugal 10 minutes then, get supernatant after, regulate pH to 8.0 with 2.0mol/L NaOH, be used for concentration and detect.
8. method according to claim 6, it is characterized in that, the described detection architecture that contains known melamine concentration is meant: get 10 centrifuge tubes that comprise melamine aptamers, nano-Au solution and PDDA solution, the melamine titer that adds variable concentrations and process pre-treatment respectively, make that content of melamine maintains 0.1-1.0 μ M in the whole detection architecture, measure behind the reaction 10min.
9. method according to claim 6, it is characterized in that, described typical curve is meant: with light absorption value A640 and the A520(OD of UV spectrophotometer measuring detection architecture at 640nm and 520nm place), and be horizontal ordinate with the melamine concentration, Δ A=A640/A520 obtains typical curve as ordinate.
10. method according to claim 6 is characterized in that, described typical curve is y=-0.002+0.883x.
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| CN104215618A (en) * | 2014-09-22 | 2014-12-17 | 上海交通大学 | Silver ion detection method based on nano-gold accumulation and dispersion quenching Rhodamine B fluorescence |
| CN104606919A (en) * | 2014-12-31 | 2015-05-13 | 华南师范大学 | Preparation method of novel nucleic acid aptamer/nano-gold modified solid phase microextraction quartz capillary tube |
| CN104897596A (en) * | 2015-05-25 | 2015-09-09 | 东华大学 | Method for detecting chiral compound based on aptamer modified nanogold |
| CN104931684A (en) * | 2015-02-28 | 2015-09-23 | 青岛科技大学 | Nano fluorescence sensor and preparation method and application thereof |
| CN104931467A (en) * | 2015-02-28 | 2015-09-23 | 青岛科技大学 | Intracellular adenosine triphosphate (ATP) fluorescence imaging method |
| CN106501203A (en) * | 2016-10-09 | 2017-03-15 | 中南大学 | A kind of insulin human detection method based on aptamer |
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| CN109115705A (en) * | 2018-08-31 | 2019-01-01 | 上海应用技术大学 | A method of utilizing cyromazine in aptamer and PDDA assemble nanometer detection of particles milk |
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| CN110567952A (en) * | 2019-09-29 | 2019-12-13 | 上海应用技术大学 | aptamer-modified nanogold-based cyromazine detection method |
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| CN103439320A (en) * | 2013-09-04 | 2013-12-11 | 青岛科技大学 | Method for determining melamine (Me) by chemiluminescence |
| CN103439320B (en) * | 2013-09-04 | 2015-06-17 | 青岛科技大学 | Method for determining melamine (Me) by chemiluminescence |
| CN104215618A (en) * | 2014-09-22 | 2014-12-17 | 上海交通大学 | Silver ion detection method based on nano-gold accumulation and dispersion quenching Rhodamine B fluorescence |
| CN104606919A (en) * | 2014-12-31 | 2015-05-13 | 华南师范大学 | Preparation method of novel nucleic acid aptamer/nano-gold modified solid phase microextraction quartz capillary tube |
| CN104931467A (en) * | 2015-02-28 | 2015-09-23 | 青岛科技大学 | Intracellular adenosine triphosphate (ATP) fluorescence imaging method |
| CN104931684A (en) * | 2015-02-28 | 2015-09-23 | 青岛科技大学 | Nano fluorescence sensor and preparation method and application thereof |
| CN104931467B (en) * | 2015-02-28 | 2018-08-21 | 青岛科技大学 | A kind of fluorescence imaging method of intracellular ATP |
| CN104897596A (en) * | 2015-05-25 | 2015-09-09 | 东华大学 | Method for detecting chiral compound based on aptamer modified nanogold |
| CN106501203A (en) * | 2016-10-09 | 2017-03-15 | 中南大学 | A kind of insulin human detection method based on aptamer |
| CN107941776A (en) * | 2018-01-03 | 2018-04-20 | 吉林大学 | The method that nucleic acid is sensitized rare earth luminous sensor detection melamine |
| CN109115705A (en) * | 2018-08-31 | 2019-01-01 | 上海应用技术大学 | A method of utilizing cyromazine in aptamer and PDDA assemble nanometer detection of particles milk |
| CN110441508A (en) * | 2019-05-06 | 2019-11-12 | 华南理工大学 | Acrylamide in Foods Rapid detection test strip based on nanogold composite coating |
| CN110441508B (en) * | 2019-05-06 | 2020-11-24 | 华南理工大学 | Test strip for rapidly detecting acrylamide in food based on nanogold composite coating |
| CN110261340A (en) * | 2019-05-24 | 2019-09-20 | 同济大学 | A kind of quick visualization analyzes the method and sensor of a variety of phthalate total amount of pollutant |
| CN110567952A (en) * | 2019-09-29 | 2019-12-13 | 上海应用技术大学 | aptamer-modified nanogold-based cyromazine detection method |
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